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The migratory ability of microglia facilitates their rapid transport to a site of injury to kill and remove pathogens. However, the effect of Treponema pallidum membrane proteins on microglia migration remains unclear. The effect of Tp47 on the migration ability and autophagy and related mechanisms were investigated using the human microglial clone 3 cell line. Tp47 inhibited microglia migration, the expression of autophagy-associated protein P62 decreased, the expression of Beclin-1 and LC3-II/LC3-I increased, and the autophagic flux increased in this process. Furthermore, autophagy was significantly inhibited, and microglial cell migration was significantly increased after neutralisation with an anti-Tp47 antibody. In addition, Tp47 significantly inhibited the expression of p-PI3K, p-AKT, and p-mTOR proteins, and the sequential activation of steps in the PI3K/AKT/mTOR pathways effectively prevented Tp47-induced autophagy. Moreover, Tp47 significantly inhibited the expression of p-FOXO1 protein and promoted FOXO1 nuclear translocation. Inhibition of FOXO1 effectively suppressed Tp47-induced activation of autophagy and inhibition of migration. Treponema pallidum membrane protein Tp47-induced autophagy and inhibited cell migration in HMC3 Cells via the PI3K/AKT/FOXO1 pathway. These data will contribute to understanding the mechanism by which T. pallidum escapes immune killing and clearance after invasion into the central nervous system., (© 2023 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

PCR can be a supplement to Treponema serological testing. However, its sensitivity is not satisfactory for blood sample testing. The aim of this study was to investigate whether pretreatment with red blood cell (RBC) lysis could enhance the yield of Treponema pallidum subsp. pallidum DNA extraction from blood. We developed and verified the efficacy of a quantitative PCR (qPCR) assay that utilizes TaqMan technology to specifically detect T. pallidum DNA by targeting the polA gene. Simulation media with 10 6 to 10 0 treponemes/mL were prepared in normal saline (NS), whole blood, plasma, and serum, and RBC lysis pretreatment was performed on a portion of whole blood. Then, blood samples drawn from 50 syphilitic rabbits were divided in parallel into five groups, labeled whole blood, whole blood/lysed RBCs, plasma, serum, and blood cells/lysed RBCs. DNA extraction and qPCR detection were performed. The detection rate and copy number were compared among different groups. The polA assay showed good linearity and an excellent amplification efficiency of 102%. In the simulated blood samples, the detection limit of the polA assay reached 1 × 10 2 treponemes/mL in whole blood/lysed RBCs, plasma, and serum. However, the detection limit was only 1 × 10 4 treponemes/mL in NS and whole blood. Among the blood samples from syphilitic rabbits, whole blood/lysed RBCs showed the best detection rate (82.0%), while the detection rate for whole blood was only 6%. The copy number of whole blood/lysed RBCs was higher than that of whole blood. RBC lysis pretreatment can significantly improve the yield of T. pallidum DNA from whole blood, and the yield is better than that from whole blood, plasma, serum, and blood cells/lysed RBCs. IMPORTANCE Syphilis is a sexually transmitted disease caused by T. pallidum that can spread into the blood. T. pallidum DNA can be detected in blood by PCR but with low sensitivity. Few studies have applied RBC lysis pretreatment to blood T. pallidum DNA extraction. This study shows that the detection limit, detection rate, and copy number of whole blood/lysed RBCs were better than those of whole blood, plasma, and serum. After RBC lysis pretreatment, the yield of low concentrations of T. pallidum DNA was improved, and the low sensitivity of blood-based T. pallidum PCR was improved. Therefore, whole blood/lysed RBCs are the ideal sample for acquiring blood T. pallidum DNA., Competing Interests: The authors declare no conflict of interest.

Aim: The present study examined the membrane location of cardiolipin antigen in treponemes. Materials & methods: The authors used different methods to disrupt the outer membrane of treponemes, detected the location of the cardiolipin antigen and analyzed the immune response in rabbits immunized with various antigens. Results: All organisms were labeled with nontreponemal antibodies on immunoelectron and fluorescence microscopy, except the citrate buffer-treated group, which is a method leading to relatively complete removal. Except for citrate buffer-treated spirochetes, all treponemes produced low-titer, nontreponemal antibodies in immunized rabbits. Conclusion: These findings indicated that the cardiolipin antigen was localized in the outer membrane of spirochetes. This study provided further evidence of the origin of nontreponemal antibodies during Treponema pallidum infection.

Vascular inflammation is a complex and multifactorial pathophysiological process that plays a crucial role in all stages of syphilis and is responsible for tissue damage. Little is known about the interactions of infiltrating immunocytes with human dermal vascular smooth muscle cells (HDVSMCs) in arterioles during the immunopathogenesis of syphilis. The Treponema pallidum subsp. pallidum membrane protein Tp47 is considered a major inducer of inflammation initiation and development. In this study, we demonstrated that Tp47 promoted the migration and adhesion of THP-1 cells to HDVSMCs. Furthermore, Tp47 increased monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) mRNA and protein expression levels in a dose- and time-dependent manner. The migration and adhesion of THP-1 cells to HDVSMCs were significantly suppressed by anti-MCP-1 and anti-ICAM-1 neutralizing antibodies, respectively. Further studies revealed that treatment of HDVSMCs with Tp47 activated the PI3K/Akt, p38 MAPK and NF-κB signalling pathways. Inhibition of PI3K/Akt, p38 MAPK and NF-κB suppressed the MCP-1 and ICAM-1 expression induced by Tp47. In addition, the migration and adhesion of THP-1 cells to Tp47-treated HDVSMCs were significantly decreased by pretreatment with PI3K/Akt, p38 MAPK and NF-κB inhibitors. These findings demonstrate that Tp47 promotes the migration and adherence of THP-1 cells to HDVSMCs by inducing MCP-1 and ICAM-1 expression, which is mediated by activation of the PI3K/Akt, p38 MAPK and NF-κB pathways. This study provides a novel potential therapeutic strategy for controlling the vascular inflammatory response in syphilis patients., (Copyright © 2019. Published by Elsevier Inc.)

Background: The highly variable tprK gene of Treponema pallidum has been acknowledged to be one of the mechanisms that causes persistent infection. Previous studies have mainly focused on the heterogeneity in tprK in propagated strains using a clone-based Sanger approach. Few studies have investigated tprK directly from clinical samples using deep sequencing., Methods/principal Findings: We conducted a comprehensive analysis of 14 primary syphilis clinical isolates of T. pallidum via next-generation sequencing to gain better insight into the profile of tprK in primary syphilis patients. Our results showed that there was a mixture of distinct sequences within each V region of tprK. Except for the predominant sequence for each V region as previously reported using the clone-based Sanger approach, there were many minor variants of all strains that were mainly observed at a frequency of 1-5%. Interestingly, the identified distinct sequences within the regions were variable in length and differed by only 3 bp or multiples of 3 bp. In addition, amino acid sequence consistency within each V region was found among the 14 strains. Among the regions, the sequence IASDGGAIKH in V1 and the sequence DVGHKKENAANVNGTVGA in V4 showed a high stability of inter-strain redundancy., Conclusions: The seven V regions of the tprK gene in primary syphilis infection demonstrated high diversity; they generally contained a high proportion sequence and numerous low-frequency minor variants, most of which are far below the detection limit of Sanger sequencing. The rampant variation in each V region was regulated by a strict gene conversion mechanism that maintained the length difference to 3 bp or multiples of 3 bp. The highly stable sequence of inter-strain redundancy may indicate that the sequences play a critical role in T. pallidum virulence. These highly stable peptides are also likely to be potential targets for vaccine development., Competing Interests: The authors have declared that no competing interests exist.

In this work, we employed real-time PCR analysis targeting tp0574 to investigate the effects of different processing procedures on the yield of T. pallidum DNA from blood to improve assay sensitivity. The T. pallidum DNA yields following red blood cell lysis pretreatment were 40.4 times greater from whole blood and 32.4 times greater from residual hematocytes than yields without pretreatment. For the simulated whole-blood experiments, the T. pallidum DNA yields from the lower layer were 2.8, 4.6, 7.3, 12.6, 15.24, 16.7, 65.1 and 73.1 times those from the upper layer following centrifugation at 500×, 1000×, 2000×, 4000×, 5000×, 7000×, 10,000× and 20,000 × g, respectively. However, the T. pallidum DNA yields from blood clots were only 1.0% at different centrifugal forces. The experiment with infected rabbit blood showed results similar to those mentioned above. In addition, sample processing time (within 48 h) and storage temperature (4 °C and 25 °C) did not affect T. pallidum DNA extraction efficiency. The T. pallidum DNA yield can be significantly improved by red blood cell lysis pretreatment and appropriate centrifugation. Furthermore, the T. pallidum DNA extraction yield is greater from whole blood or residual hematocytes from anti-coagulated blood than from plasma, serum or blood clots., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: The involvement of inflammasome activation and macrophage polarization during the process of syphilis infection remains unknown. In this study, A series of experiments were performed using human macrophages to research the role of NLRP3 inflammasome regulation in interleukin (IL)-1β production and its influence on macrophage polarization triggered by T. pallidum., Results: The results showed that in M0 macrophages treated with T. pallidum, the M1-associated markers inducible nitric oxide synthase (iNOS), IL-1β and TNF-α were upregulated, and the M2-associated markers CD206 and IL-10 were downregulated. In addition, we observed NLRP3 inflammasome activation and IL-1β secretion in T. pallidum-treated macrophages, and the observed production of IL-1β occurred in a dose- and time-dependent manner. Moreover, the secretion of IL-1β by macrophages after T. pallidum treatment was notably reduced by anti-NLRP3 siRNA and caspase-1 inhibitor treatment. NAC, KCl, and CA074-ME treatment also suppressed IL-1β release from T. pallidum-treated macrophages., Conclusions: These findings showed that T. pallidum induces M0 macrophages to undergo M1 macrophage polarization and elevate IL-1β secretion through NLRP3. Moreover, the process of NLRP3 inflammasome activation and IL-1β production in macrophages in response to T. pallidum infection involves K + efflux, mitochondrial ROS production and cathepsin release. This study provides a new insight into the innate immune response to T. pallidum infection.

The origin of nontreponemal antibodies during syphilis infection is hotly debated. Here, we analyzed the immune response in rabbits immunized with various antigens. Inactivated treponemes elicited the production of low-titer nontreponemal antibodies in some rabbits. Cardiolipin combined with bovine serum albumin also induced anticardiolipin antibody production. These findings indicate that Treponema pallidum contained a cardiolipin antigen with weak immunogenicity. However, active T. pallidum induced higher nontreponemal antibody production with strong immunogenicity at an earlier time point, and the antibody titer was consecutive, suggesting the high nontreponemal antibody titer resulted from the combined effects of both the T. pallidum cardiolipin antigen and the damaged host-cell cardiolipin antigen during syphilis infection, the latter of which plays a major role in the induction of nontreponemal antibody production. Our study provides direct animal evidence of the origin of nontreponemal antibodies during T. pallidum infection.

The polarization of macrophages and the molecular mechanism involved during the early process of syphilis infection remain unknown. This study was conducted to explore the influence of Treponema pallidum (T. pallidum) treatment on macrophage polarization and the Akt-mTOR-NFκB signaling pathway mechanism involved in this process. M0 macrophages derived from the phorbol-12-myristate-13-acetate-induced human acute monocytic leukemia cell line THP-1 were cultured with T. pallidum. T. pallidum induced inflammatory cytokine (IL-1β and TNF-α) expression in a dose- and time-dependent manner. However IL-10 cytokine expression decreased at the mRNA and protein levels. Additionally, the expression of the M1 surface marker iNOS was up-regulated with incubation time, and the expression of the M2 surface marker CD206 was low (vs. PBS treated macrophages, P < 0.001) and did not fluctuate over 12 h. Further studies revealed that Akt-mTOR-NFκB pathway proteins, including p-Akt, p-mTOR, p-S6, p-p65, and p-IκBα, were significantly higher in the T. pallidum-treated macrophages than in the PBS-treated macrophages (P < 0.05). In addition, inflammatory cytokine expression was suppressed in T. pallidum-induced M1 macrophages pretreated with LY294002 (an Akt-specific inhibitor) or PDTC (an NF-κB inhibitor), while inflammatory cytokine levels increased in T. pallidum-induced M1 macrophages pretreated with rapamycin (an mTOR inhibitor). These findings revealed that T. pallidum promotes the macrophage transition to pro-inflammatory M1 macrophages in vitro. The present study also provides evidence that Akt, mTOR and NF-κB pathway activation in T. pallidum stimulates M1 macrophages. This study provides novel insights into the innate immune response to T. pallidum infection., (Copyright © 2018. Published by Elsevier B.V.)

This study aimed to determine whether a serological response could predict the normalization of cerebrospinal fluid (CSF) abnormalities at 6 months after treatment in human immunodeficiency virus (HIV)-negative neurosyphilis patients. A total of 123 neurosyphilis patients were recruited at baseline, 58 of these patients undergoing treatment, repeated CSF examinations and serological tests for syphilis at 6 months after treatment were included in the follow-up study. Before treatment, the CSF rapid plasma reagin (RPR) titer, CSF Treponema pallidum particle agglutination (TPPA) titer, CSF leukocyte count, and CSF protein concentration were correlated with both serum RPR and TPPA titers. At 6 months after treatment, 28 and nine patients achieved serological responses of RPR and TPPA tests, respectively. The sensitivities of the serological response of RPR and TPPA tests for identifying the normalization of CSF abnormalities were 60.0∼83.3% and 17.1~22.2%, respectively; and 75.0∼91.3% of patients showing serological response of RPR test also achieved CSF normalization, suggesting that the serological response could predict CSF normalization to some degree. Particularly, in patients with ≥8-fold decreases in the serum RPR titer, the CSF RPR, CSF leukocyte count, and CSF protein concentration had normalized, and follow-up lumbar puncture could be reduced considering the resolution of neurological symptoms.

Treponema pallidum ssp. pallidum (T. pallidum), the causative agent of the sexually transmitted disease syphilis, is an uncultivatable human pathogen. The geographical differences in T. pallidum genomes leading to differences in pathogenicity are not yet understood. Presently, twelve T. pallidum genomes are available to the public, all of which are American in origin and often co-infect patients with human immunodeficiency virus (HIV). In this study, we examined the T. pallidum subsp. pallidum strain Amoy, a syphilis pathogen found in Xiamen, China. We sequenced its genome using Illumina next-generation sequencing technology and obtained a nearly (98.83%) complete genome of approximately 1.12 Mbps. The new genome shows good synteny with its five T. pallidum sibling strains (Nichols, SS14, Mexico A, DAL-1, and Chicago), among which SS14 is the strain closest to the Amoy strain. Compared with strain SS14, the Amoy strain possesses four uncharacterized strain-specific genes and is likely missing six genes, including a gene encoding the TPR domain protein, which may partially account for the comparatively low virulence and toxicity of the Amoy strain in animal infection. Notably, we did not detect the 23S rRNA A2058G/A2059G mutation in the Amoy strain, which likely explains the sensitivity of Amoy strain to macrolides. The results of this study will lead to a better understanding of the pathogenesis of syphilis and the geographical distribution of T. pallidum genotypes.

Background: The rabbit infectivity test (RIT) was previously described as a highly-sensitive method for clinically detecting Treponema pallidum. But our primary study indicated this result may have changed in current antibiotics era., Methods: By inoculating rabbits testis with cerebrospinal fluid (CSF) (n=63) and exudate from hard chancre lesions (n=13), we re-evaluated the sensitivity of RIT in modern era. All isolated T. pallidum strains from the RIT were performed for the strain type based on "CDC subtype/tp0548" method. Chi-square and Fisher's exact tests were used to determine the statistical significance of differences across data sets., Results: Result indicated that 2 of 63 CSF (2/63, 3.17%) and 5 of 13 lesion exudate samples (5/13, 38.47%) were positive in the RIT, with a much longer time to detection for CSF samples. Only 1 of 28 samples from patients who admitted treatment with antibiotics prior to clinical exam was positive in the RIT; while 6 of 48 patients, who admitted no recent exposure to antibiotics or was unclear about the medical history, were positive in RIT. DNA sequence analysis revealed 6 strains of 14d/f subtype and one strain of 14a/f subtype., Conclusions: In conclusions, RIT is no longer a highly sensitive method for detecting T. pallidum in clinical samples as before, and is not inadequately considered to be a reference method for measuring the sensitivity of other new methods, such as the PCR. These data represent the first reexamination of the sensitivity of RIT in the post-antibiotic era with a large clinical sample., (Copyright © 2016. Published by Elsevier B.V.)

Background: No gold standard currently exists for the diagnosis of general paresis (GP), thus often resulting in unnecessarily delayed therapeutic decision., Methods: A retrospective chart review was performed for 85 inpatients with GP in Zhongshan Hospital, Medical College of Xiamen University, and the characteristics of their clinical profiles, serum and cerebrospinal fluid (CSF) examinations, neuroimaging examination, and electroencephalogram (EEG) data were analyzed., Results: Among the 85 GP patients, the clinical symptoms that were frequently observed upon admission included a variety of psychiatric-behavioral symptoms and varying degrees of cognitive impairment. All of the patients had positive serum Treponema pallidum particle agglutination (TPPA) assays, 96.47% of the patients had positive CSF TPPA assays, and 41.18% of the patients had both CSF pleocytosis and elevated CSF protein levels. Focal atrophy in one cerebral region or in multiple regions was evident in neuroimages. The EEG data primarily showed slightly abnormal EEG activity., Conclusion: These results demonstrate the complexity of the clinical characteristics of GP and highlight the importance of early diagnosis., (Copyright © 2014 Elsevier B.V. All rights reserved.)